Early life environmental tobacco smoke (ETS) exposure is associated with detrimental outcomes on neurodevelopment, including Attention-Deficit / Hyperactivity Disorder (ADHD), but the mechanisms are unclear. Epi-genetic changes induced by ETS exposure may underlie these associations. Our long-term goal is to utilize DNA methylation profiles to identify children at risk for developing adverse neurodevelopmental outcomes. The objective is to uncover the epigenetic mechanism linking ETS exposure in early life to phenotypes associated with ADHD. We hypothesize that early life ETS exposure induces persistent and targeted functional changes in DNA methylation that associate with neurodevelopmental phenotypes. The rationale for is that defining the repertoire of ETS-induced functional DNA methylation changes will offer exciting new opportunities for risk stratification and early interventions to reduce and prevent adverse behavioral and cognitive outcomes of ETS exposure. Our aims are as follows: 1) Identify ETS-related methylation targets. We will test the hypothesis that early life ETS exposure in rats will induce measurable shifts in methylation at loci relevant to neurobehav-loral outcomes that are detectable in both brain and blood. Whole genome bisulfite sequencing will identify ETS-vulnerable loci with validation by pyrosequencing. 2) Identify ETS-altered methylation-expression relationships in frontal cortex. We hypothesize that early life ETS exposure in rats induces changes in frontal cortex gene expression at ADHD-relevant genes. Whole transcriptome profiling will identify these changes. We will also evaluate methylation expression relationships from the in vivo results and in an in vitro model of neuronal differentiation. 3) Determine if DNA methylation varies with ETS dose in humans. Prenatal and peri-natal cotinine levels and DNA methylation in children, from bisulfite pyrosequencing at ADHD-related genes and candidate loci from the above aims, will determine if methylation varies with exposure. The proposed research is expected to reveal ETS-related epigenetically vulnerable genes that mechanistically explain the link between ADHD and exposure to ETS and that may serve as biomarkers of past exposure.